On Oct 16, 11:08 am, "Kotredes, Kevin"
<Kevin_Kotre... from DFCI.HARVARD.EDU> wrote:
> I'm interested in learning and practicing the procedure to prepare zebrafish
> sperm for cryopreservation and later use for in vitro fertilization. Does
> anybody have a useful protocol they are willing to share or a resource that they
> can suggest? Thank you very much!
>> Kevin P Kotredes
> Research Technician
> Look Zebrafish Lab
> Jimmy Fund Building, RM G03
> Dana-Farber Cancer Institute
> Phone: 617-632-3545
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Kevin,
I'm trying to start up sperm cryopreservation at my lab as well.
Multiple sperm freeze protocols can be found at the ZIRC site:
http://zebrafish.org/zirc/documents/protocols.php
Also, there is a protocol in the Christiane Nusslein-Volhard/Ralf Dahm
Zfish book:
http://books.google.com/books?id=zS-5CKOjCIgC&pg=PA30&dq=zebrafish+sperm+cryopreservation
The Draper protocol appears to be the most widely used (e.g. at ZIRC,
in TILLING at Moens Lab, etc.) and is well written/easy to follow.
....however, I am following it and thus far have been having a really
difficult time getting the 1-3ul of sperm that the protocol
recommends. If anyone reading this can recommend or describe a
squeezing technique that can produce larger volumes, please please
take the time to post some advice. I have tried everything I can
think of, variety of forceps, fingers, variety of pressures, variety
of anatomical regions, sexy music, etc. Anyone? Please........